Protein self-assembly following in situ expression in artificial and mammalian cells.

The self-assembly of proteins has been widely studied in controlled in vitro conditions, and more recently in biological environments. The self-assembly of proteins in biology can be a feature of the pathogenesis of protein condensation disease, or can occur during normal physiological function, for example during the formation of intracellular non-membrane bound organelles. To determine the mechanisms for the assembly process fully, controlled in vitro experiments using purified protein solutions are often required. However, making direct connections between insights gathered from controlled experiments and those in complex biological environments remains a challenge. Using the P23T mutant of human γD-crystallin, a protein associated with congenital cataract, we have demonstrated that the equilibrium solubility boundary and solution behavior measured using phase diagrams of purified protein solutions is consistent with the assembly of the protein expressed in cell-free expression medium in artificial cells (without fluorescent labelling) and condensates formed in mammalian cells, thereby directly connecting in vitro measurements with those performed under physiological conditions.

Adding glycolipid functionality to model membranes--phase behaviour of a synthetic glycolipid in a phospholipid membrane.

Glycolipid phase behaviour is less well understood than for many phospholipids, but due to their structural and functional diversity, glycolipids represent an important group of amphiphiles from which biological function is derived. Here we have incorporated a synthetic glycolipid in binary mixtures with DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) into giant unilamellar vesicles (GUVs) at biologically relevant concentrations and observed the phase behaviour of the lipid mixtures for a range of glycolipid concentrations. At low concentrations, the glycolipid is fully dispersed in the GUV membrane. At glycolipid molar concentrations above 10%, the formation of lipid tubules is observed, and is consistent with the formation of a columnar lipid phase. Lipid tubules are observed in aqueous and oil solvents, suggesting that both hexagonal and inverted hexagonal lipid arrangements can be formed. This work may offer insights into the biological function of glycolipids and the challenges in formulating them for use in industrial applications.